For many gastrointestinal diseases, a promising new treatment concept uses engineered microbes to synthesize therapeutics in situ. To implement this approach, synthesis must be reliable, controllable, and adjustable. With engineered bacteria, this can be accomplished by genetically regulating expression of proteins required to create the therapeutic via promoters controlled by ligand-inducible transcription factors. Many natural transcription factors have been characterized that respond to their external environments via a wide range of small molecule signals. However, the majority of synthetic gene circuits have been built with just a handful of components. To expand these capabilities, we need new ways to construct, tune, and control transcriptional regulatory networks. Ideally, these methods will provide ?plug-and-play? components that can be used to build complex circuits, and will respond to signaling molecules incorporated into the patient's diet. To that end we will construct and integrate two complementary libraries, comprising: (1) engineered transcription factors that respond to a large range of edible chemical inducers; and (2) highly responsive and tunable promoters that are regulated by the engineered transcription factors. Our key innovation is the manner in which we employ chimeric transcription factors. These modular proteins each contain a ligand-binding domain (LBD) and a DNA-binding domain (DBD) that can be mixed and matched to create new functionalities. Notably, chimeras with the same DBD will independently and simultaneously regulate the same promoter via a single operator site. In this manner, two-input logic gating arises from combining two chimeras that respond to different ligands; multi-input gating arises from the simultaneous use of three or more chimeras. In Aim 1, we will engineer AND gates from chimeric transcription repressors and OR gates from chimeric transcription activators. Our preliminary data show that at least four chimeric repressors can be co- expressed to create a predictable AND gate. Additional protein engineering will be used to tune the ligand concentration that is required for physiological response. In Aim 2, we will integrate the chimeric transcription factors with novel hybrid promoters engineered to have tunable -10 and -35 sites. This will allow us to specify both the leakiness of a promoter (in the absence of inducer) and its maximal output (when fully induced). We will use the output from these circuits to develop a mathematical model that will predict the capacity and performance of future chimera-based circuits. Finally, in Aim 3, we will test our hypothesis that chimeric transcription factors can be used to reliably increase the complexity and tunability of synthetic gene circuits. To that end, we will construct proof-of-principle systems that use chimeric transcription factors to couple various circuit modules, such as pulse generators and genetic oscillators. Overall, the proposed work will greatly expand the components available for engineering complex synthetic gene circuits for use in therapeutic microbes that respond to chemical signals incorporated into patient diet.